白色念珠菌烯醇化酶免疫磁珠定量检测方法的建立

Development of an immunomagnetic bead assay for quantitative detection enolase of Candida albicans

目的侵袭性念珠菌病临床致死率较高,而白色念珠菌为最常见致病菌。建立定量检测白色念珠菌烯醇化酶（enolase,Eno）的免疫磁珠方法,提高侵袭性念珠菌感染的实验室诊断水平。方法将白色念珠菌Eno单抗耦联至磁性微珠上,以辣根过氧化物酶标记羊抗Eno作为检测抗体,利用重组白色念珠菌Eno蛋白作为标准品评价该方法的精密度、特异性、线性范围、最低检测下限等性能指标。用建立的免疫磁珠方法检测不同真菌培养上清中Eno水平。结果 Eno浓度为25 ng/mL和5 ng/mL时,批内和批间精密度分别为4.54%、5.87%和5.26%、8.82%。最低检出下限为0.5 ng/mL,线性范围为0.5~50 ng/mL。白色念珠菌37℃培养24 h的上清中Eno含量为3.89 ng/mL,培养120 h的Eno水平上升至37.89 ng/mL,Eno水平与白色念珠菌菌丝含量呈正相关。该方法与近平滑念珠菌有微弱交叉反应,与热带念珠菌、光滑念珠菌、季也蒙念珠菌、新型隐球菌和酿酒酵母均无交叉反应。结论成功建立了定量检测白色念珠菌Eno的免疫磁珠法,与前期建立的定性ELISA相比,免疫磁珠法更为简便、准确、敏感,在侵袭性念珠菌感染研究中具有潜在的应用价值。

Objective To develop an immunomagnetic bead（ IMB） assay for quantitative detection enolase（ Eno） of Candida albicans,and to improve the diagonosis of invasive candidiasis.Methods The immunomagnetic bead was prepared by conjugating with Anti Eno of Candida albicans monoclonal antibody.HRP-conjugated goat polyclonal antibody against Candida albicans Eno was employed as detecting antibody.The performance parameters of the IMB assay including precision,specificity,linear range and limit of detection were verified by using recombinant Candida albicans Eno.Then the developed assay was applied to determine Eno levels in supernatant of pathogenic fungi cultures.Results The intra and inter-coefficient of variation was 4.54%,5.87% and5.26%,8.82% at the concentration of 25 ng /mL and 5 ng /mL,respectively.The limit of detection was 0.5 ng /mL.The linear range was（ 0.5- 50） ng /mL.The level of Eno in Candida albicans culture after incubated in 37 ℃ for 24 h was 3.89 ng /mL and gradually increased to 37.89 ng /mL at 120 h.There was a positive correlation between the level of Eno and growth hyphae of Candida albicans.There was weak cross reaction with Candida parapsilosis and no cross reaction with Candida tropicalis,Candida guilliermondii,Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae.Conclusion An IMB assay for quantitative detection Eno of Candida albicans was developed,which was more sensitive,rapid and reliable than previous qualitative ELISA.The IMB assay has the potential to be applied to the research in invasive candidasis.